Primary skeletal muscle cell lines have been established from muscle biopsies from Pima Indians with varying in vivo insulin sensitivities. Muscle biopsies were performed in conjunction with a hyperinsulinemic euglycemic clamp to measure in vivo insulin sensitivity. The cells derived from these biopsies maintained the ability to fuse and the resulting myotubes morphologically resemble myocytes. Antibodies directed against a-actin and myosin detect both of these proteins in fixed cells indicating cultures contain cells of muscle lineage. Muscle-specific creatine kinase is also expressed by these cells. RNA encoding GLUT4, the skeletal muscle glucose transporter was detected as was RNA encoding MYOD, MYF5, and MYOGENNIN, muscle-specific differentiation factors. The myoblasts also retain differing capacities for non-oxidative glucose metabolism. Glycogen is produced in these cells in a time dependent manner. Glycogen production also increases in response to increasing concentrations of insulin. Glycogen production was also measured in these cell lines in response to insulin-stimulation. The variation in glycogen production between cell lines correlated well with in vivo measures of insulin action as measured by the hyperinsulinemic euglycemic clamp indicating the retention of insulin resistance in vitro. This observation was extended by measuring glucose transport and the cellular localization of glucose transporters with insulin stimulation. The correlation between glucose to glycogen is mirrored by glucose transport in primary skeletal muscle cultures.